{Reference Type}: Journal Article
{Title}: Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.
{Author}: Kang T;Budhraja R;Kim J;Joshi N;Garapati K;Pandey A;
{Journal}: Cell Rep Methods
{Volume}: 4
{Issue}: 4
{Year}: 2024 Apr 22
暂无{DOI}: 10.1016/j.crmeth.2024.100744
{Abstract}: A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.